There is extensive experience in the immobilization of biologically active molecules to surfaces for research and clinical use. Techniques for enzyme immobilization, adsorption of affinity reagents such as antibodies to surfaces for use in separation procedures and diagnostic assays, and covalent attachment of such reagents or enzymes to surfaces for industrial and medical use are known. More recently, medical devices which incorporate biologically active molecules as functional components have been employed in extracorporeal treatments wherein the treated fluids from the patient will be used directly for administration. It is not practical to treat the fluid which is exiting the device so as to assure freedom from contamination that might have been acquired in transit. Therefore, it is essential that the device through which the fluid is passed itself be sterile.
Ordinary techniques, such as autoclaving and irradiation for sterilizing medical equipment are problematic because these procedures would inactivate the biologically active molecule that is an integral part of the apparatus. Furthermore, the biologically active molecule may be destabilized by the process of coupling it to the surface, and techniques are needed to provide stability with respect to this aspect, as well.
There are many reports of coupling active biological materials to solid support. For example, Larsson, P. H., et al., J Immunol Meth (1989) 116:293-298, describe a method for covalent attachment of antibodies to polystyrene dishes wherein the resulting covalently derivatized dishes can then be used in cell depletion procedures in a panning process. U.S. Pat. No. 4,933,410, issued 12 Jun. 1990, also describes a method for derivatizing polystyrene surface so as to acquire the capability covalently to bind active proteins such as antibodies and their fragments, protein A, complexing agents, and other substituents. Derivatization of a polystyrene surface through covalent linkage to antibodies or their fragments is also described by Peterman, J. H., et al., J Immunol Meth (1988) 111:271-275; Chu, V. P., et al., J Ap Polymer Sci (1987) 34:1917-1924, describes covalent derivatization of polystyrene surfaces for immunoassay protocols. In addition, enzyme immobilization using covalent linkages was described by Rossi, V., et al., Int J Art Org (1981) 4:102-107. The immobilization of arginase, described in this article, did not include procedures for sterilization; however, in a subsequent article, Callegaro, L., et al., Int J Art Org (1983) 6:19-96, the same group describes the use of fibers containing L-asparaginase which are sterilized using .gamma.-irradiation, under conditions, not specified, which apparently did not completely inactivate the enzyme. None of the other references cited above address the problem of sterilization of the activated surfaces.
The difficulties of using ionizing radiation to sterilize solid surfaces containing biologically active materials was disclosed with respect to Factor XIII grafted onto collagen films by Blanchy, B. G., et al., J Biomed Mat Res (1986) 20:469-479, which describes the sensitivity of Factor XIII to .gamma.-radiation. Some improvement was effected by utilizing electron beam irradiation.
The problem of providing sterile activated surfaces is addressed directly by a number of publications. A general summary of approaches to sterilization of medical devices or biosensors is provided by Cesar, E. Y., et al., "Biosensors in Artificial Organs," ASAIO Trans (1987) 33:840-845. Some of the literature reviewed in this publication includes a description of the use of antibiotics to sterilize a glucose sensor (Kondo, T. et al., Diabetes Care (1982) 5:218:221); the use of propylene oxide to sterilize immunoadsorbents (Sato, H., et al., Int J Art Org (1985) 8:109-114), and ionizing radiation is described by Sato, H., et al., Int J Art Org (1986) 9:131-136. In the last paper cited, it was noted that losses were observed in adsorptive capacity of the conjugated antibodies even after freeze-drying from a 2% mannitol solution. Woolston, J., in Med Device Technol (1990) 1:24-31, gives a general survey of publications on the irradiation sterilization of medical devices which contain biologically active proteins.
Because ionizing radiation is known to effect chemical reactions in the target, the nature of these reactions has been explored with respect to peptides and proteins in a series of articles by Davies, K. J. A., and others in J Biol Chem (1987) 20:9895-9901, 9902-9907, 9908-9913. In this series of papers, it is noted that chemical scavengers for oxygen radicals, including t-butyl alcohol, isopropyl alcohol, mannitol, and urate could protect irradiated molecules from chemical degradation due to the products of radiation. This general picture of the chemical effects of irradiation is also described in a review article by Garrison, W. M., Chem Rev (1987) 87:381-398.
The construction of a surface with covalently attached monoclonal antibody and sterilized for medical use is described by Morecki, S., et al., J Biol Resp Modif (1990) 9:463-474. As is generally the case in providing surfaces with biologically active materials which are supplied in dried form, the surface is blocked with albumin before drying to stabilize the biological material during the drying process and in the dried state. The dried surfaces were reported to be sterilized using electron beam irradiation. No comment is made as to the effect of the irradiation sterilization process on the effectiveness of the resulting surface in adsorbing target cell subsets.
Taken together, the foregoing reports indicate that sterilization of surfaces to which biologically active materials, especially proteins, are coupled, gives unpredictable results with respect to maintaining the activity or binding capacity of the coupled active factors. There is clearly a need for a protocol that can assure the integrity of the biocapacity of the surface when irradiation sterilization is employed to assure freedom from contamination.